Lactic dehydrogenase (LDH) appears in human plasma and serum as five isoenzymes. These enzymes are tetrameric proteins composed of two subunit types `M` (prevalent in skeletal Muscle) and `H` (prevalent in the Heart). The isoenzymes were originally identified by electrophoretic separation and are conventionally referred to as LD-1, LD-2, LD-3, LD-4 and LD-5 based on their relative migration towards the anode, LD 1 being the fastest. Their corresponding subunit compositions are H.sub.4, H.sub.3 M, H.sub.2 M.sub.2, HM.sub.3 and M.sub.4 respectively.
The proportional amount of each isoenzyme varies considerably between organs, but within any given organ is relatively constant, such that each organ has a characteristic isoenzyme profile. For example, heart and erythrocytes have high proportions of LD-1 and LD-2, liver and skeletal muscle contain predominantly LD-5, and lung, kidney and brain contain mixtures in which LD-2, LD-3 and LD-4 may predominate to varying degrees. Leakage of these enzymes from a diseased or damaged organ will result in an elevation of total serum LDH, and further characterization of the serum isoenzyme composition can aid in identifying the organ responsible for this leakage. Thus, determinations of serum LDH and its isoenzyme composition are useful in the diagnosis of a variety of disease states. Diagnosis of suspected myocardial infarction is the most frequent application of LDH isoenzyme determinations. In these cases, an increase in the LD-1 isoenzyme relative to the other forms is specific and confirmatory for myocardial damage since the heart contains the largest proportion of the LD-1 enzyme.
Electrophoresis was the first method used to separate the LDH isoenzymes, and is still widely used to obtain complete isoenzyme profiles. This method has several disadvantages in that it requires multiple and lengthy steps, and expensive equipment. Separations using ion exchange columns have also been developed, but also have disadvantages.
An alternative approach to the analysis of LDH isoenzymes has been to selectively inhibit or denature some of the isoenzymes and so quantitate the relative proportion of those resistant to the treatment. Examples include: the use of urea, heat and high pH to progressively inactivate those isoenzymes containing the most `M` subunits; the use of high substrate levels (pyruvate or lactate) to selectively inhibit the `H` subunit; and the use of .alpha.-ketobutyric acid as a substrate specific for the H subunit.
U.S. Pat. No. 4,250,255 describes an approach where the total LDH activity is first measured and then the isoenzymes are selectively inhibited by treating the sample with an ionic amphiphile. The treated sample is then measured for enzyme activity and subtracted from the first measurement. The difference is the activity of the isoenzyme.
The subject invention provides an alternative to the above approaches and is a highly specific method for the determination of LD-1 isoenzyme in biological fluids.